antioxidants Review The Role of Methionine Sulfoxide Reductases in Oxidative Stress Tolerance and Virulence of Staphylococcus aureus and Other Bacteria Vineet K. Singh 1,* , Kuldeep Singh 2 and Kyle Baum 1 1 Department of Microbiology and Immunology, A.T. Still University of Health Sciences, Kirksville, MO 63501, USA; [email protected] 2 Mayo Clinic, Rochester, MN 53905, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-660-626-2474; Fax: +1-660-626-2523 Received: 31 August 2018; Accepted: 26 September 2018; Published: 28 September 2018 Abstract: Methionine sulfoxide reductases (MSRA1 and MSRB) are proteins overproduced in Staphylococcus aureus during exposure with cell wall-active antibiotics. Later studies identified the presence of two additional MSRA proteins (MSRA2 and MSRA3) in S. aureus. These MSR proteins have been characterized in many other bacteria as well. This review provides the current knowledge about the conditions and regulatory network that mimic the expression of these MSR encoding genes and their role in defense from oxidative stress and virulence. Keywords: MSRA; MSRB; oxidative stress; virulence 1. Methionine Sulfoxide Reductases The presence of reactive oxygen species (ROS) is potentially damaging to all cellular macromolecules. Oxidizing agents, such as hydrogen peroxide (H2O2), superoxides, and hydroxyl radicals, oxidize the sulfur atom of methionine residues, resulting in methionine sulfoxide (MetO) that typically leads to loss of protein function [1,2]. In 1981, an enzyme capable of reducing protein-bound methionine sulfoxide was identified [3,4]. These oxidized MetO residues are reduced back to methionine by methionine sulfoxide reductase (MSR) enzymes that restore normal protein functions [5,6]. Oxidation of methionine results in two stereoisomeric forms of MetO (R-MetO and S-MetO), which are reduced by two different MSR enzymes (MSRA and MSRB). MSRA is specific to S-MetO, and MSRB is specific to R-MetO [7–10]. The MetO/Met, MSRA/B-mediated oxidation and reduction of methionine residues are thus an important antioxidant mechanism [11], and methionine is no longer needed just for protein initiation [12]. 2. MSRA and MSRB Enzymes in Staphylococcus aureus and Other Bacteria In the S. aureus chromosome, there are three MSRA genes (MSRA1, MSRA2 and MSRA3) and one MSRB gene. These four MSR genes are expressed from three different promoters. The MSRA1 and MSRB genes are co-expressed as part of a polycistronic message, and the MSRA2 and MSRA3 genes are expressed independently from their respective promoters [13]. In bacteria, most species contain at least one copy of each of the MSRA and MSRB genes. However, similar to S. aureus, multiple copies of MSR encoding genes have been identified in many bacteria. Vibrio cholera contains two MSRA and three MSRB genes distributed in its two chromosomes. Rhizobium meliloti contains three MSRA and three MSRB genes with one copy of each found on a plasmid [14,15]. In addition, there is no set pattern in terms of the genetic organization of MSR genes across species. In some bacterial species, e.g., Escherichia coli, Mycobacterium tuberculosis, Bordetella pertussis, Pseudomonas aeruginosa, Salmonella Antioxidants 2018, 7, 128; doi:10.3390/antiox7100128 www.mdpi.com/journal/antioxidants Antioxidants 2018, 7, 128 2 of 10 Typhimurium, and S. aureus, MSRA and MSRB genes are two separate entities. In other bacterial species, e.g., Bacillus subtilis and S. aureus, the MSRA and MSRB genes are found adjacent to each other and are co-transcribed. Interestingly, in some other species, e.g., Neisseria gonorrhoeae, Neisseria meningitidis, and Helicobacter pylori, the MSRA and MSRB genes are fused and translated as separate domains within a single larger polypeptide [7,16]. 3. Methionine and MSR Enzymes as Part of an Antioxidant System While all amino acid residues can be oxidized, their sensitivity to oxidation is variable [17]. Methionine has a much higher propensity for oxidation than other amino acids. It is present in high concentrations on the surface of certain proteins [17] and can be oxidized without affecting the functionality of proteins. In the case of E. coli glutamine synthase, eight of the 16 methionine residues distant to the catalytic site could be oxidized without affecting activity [17]. Similar results were also seen in another E. coli protein, GroEL [18]. This protein remained fully functional even when all of its 23 methionine residues were oxidized after exposure to 15 mM H2O2 for three hours. It was only when an even higher H2O2 concentration was used, causing the oxidation of cysteines and tyrosines, that the GroEL chaperonin activity was eventually reduced [18]. The scavenging ability of methionines was further exemplified when about 40% of methionine residues in E. coli cells were substituted with norleucine, a carbon analog of methionine in which sulfur is replaced with a methylene group. These norleucine-substituted bacterial cells were more susceptible to killing by hypochlorite and peroxide [19]. Several proteins from the human gastric pathogen H. pylori were shown to interact with MSR enzymes. Many of these proteins (e.g., GroEL, catalase, and recombinase) are significantly more methionine rich than other bacterial proteins and, in all likelihood, are oxidized under oxidative stress and salvaged by the activity of MSR enzymes [20]. Thus, MSR proteins not only repair oxidative damage to methionine residues through the oxidation/reduction cycle but also serve as scavengers of ROS and protect cells from more widespread oxidative damage [11,17]. 4. Environmental Impact on MSR Expression In S. aureus, MSRA1 and MSRB proteins were first identified as proteins overproduced in response to the presence of cell wall-active antibiotics [21]. Later, the expression of MSRA1 and MSRB genes were determined to be induced at the transcriptional level and specifically in response to cell wall-active antibiotics [22,23]. Using a promoter-lacZ fusion, it was shown that none of the S. aureus MSR genes were induced under conditions of oxidative stress [24]. Cell wall-active antibiotics only induced the expression of the MSRA1/MSRB locus and had no effect on the expression of MSRA2 or MSRA3 genes [24]. A high salt concentration caused some induction of MSRA3 but not of the other S. aureus MSR genes [24]. The expression of MSRA2 and MSRA3 genes was very low, and both were expressed higher during the early exponential phase of bacterial growth [24]. The expression of the MSRA1/MSRB locus occurs at a much higher level than the expression of MSRA2 and MSRA3 and is most expressed during the late log and stationary phases of growth [24]. Surprisingly, cell wall-active antibiotics induce the expression of MSRA1 and MSRB genes in S. aureus, but these MSR enzymes appear to play no direct role in defense from these antibiotics [13]. It is speculated that cell wall-active antibiotics destabilize the cell wall and allow the oxidants easy access to cellular macromolecules. MSRA1 and MSRB might be needed at higher concentrations during exposure of S. aureus to the cell wall inhibitors. When the role of SigmaB (stress responsive sigma factor) was investigated, it enhanced, but was not required for, the expression of the S. aureus MSRA1/MSRB locus [25,26]. In addition, induction of the MSRA1/MSRB locus by cell wall-active antibiotics was inhibited by glycerol monolaurate that interfered with signal transduction pathways. This result led to the speculation that an unidentified signal transduction pathway might be involved in the induced expression of MSRA1/MSRB in the presence of cell wall-active antibiotics [26]. More recently, a higher level of expression of MSRA2 gene was observed in S. aureus after sunlight exposure in both oxic and anoxic conditions. Thus, it has been Antioxidants 2018, 7, 128 3 of 10 speculated that MSRA probably defends the bacteria from oxygen-dependent and oxygen-independent photostresses [27]. Expression of MSR genes in other pathogenic species has also been investigated. Similar to S. aureus MSRA1/MSRB, in H. pylori [20], E. coli [28], and B. subtilis [29] the late log and stationary phase cultures of these bacteria had the highest MSR activity. In addition to MSRA1/MSRB induction by cell wall-active antibiotics, piplartine, a biologically active alkaloid from peppers [30], showed enhanced MSR expression in the pathogenic parasite, Trypanosoma cruzi [31]. There is no doubt that MSRs protect cells from oxidative stress, but there are only a handful of species where oxidative stress conditions have been shown to induce the expression of MSR genes. In Streptococcus gordonii,H2O2 induced expression of the MSRA gene [32]. Oxidative stress conditions induced the expression of MSRA in the xenobiotics-degrading bacterium Ochrobactrum anthropic [33]. The expression of MSRA gene in plant bacterium Xanthomonas campestris pv. phaseoli was highly induced by exposure to oxidants and N-ethylmaleimide [34]. In P. aeruginosa,H2O2, paraquat, or sodium hypochlorite had no effect on MSRA1 expression, but expression of MSRB was induced by sodium hypochlorite [35]. Oxidative stress conditions caused transcriptional upregulation of MSR in H. pylori [36]. In addition to oxidative stress, a slight upshift in pH (from 6.2 to 7.3) also led to increased expression of MSRA in S. gordonii [37]. Finally, the B. subtilis MSRA/MSRB operon was induced by paraquat but not by H2O2 [38]. 5. Cellular Control of MSR Expression In S. aureus and B. subtilis, the expression of MSRA and MSRB genes appears to be under the control of SigmaA [26,38]. In B. subtilis, a transcriptional regulator, Spx, has a positive impact on the expression of MSRA/MSRB genes [38]. Further, paraquat induces expression of MSRA/MSRB in B. subtilis, and this phenomenon is seen even in Spx-deficient cells, suggesting that some unidentified factors also play a role [38]. YjbH acts as a post-transcriptional negative regulator of global oxidative/thiol stress in B. subtilis [39], binding and stabilizing Spx [40]; thus, YjbH may be indirectly involved in the regulation of MSRA/MSRB expression in this bacterium.